Expression of protein in mammalian cells.

Frank O. Fackelmayer Frank.Fackelmayer at uni-konstanz.de
Fri May 26 02:10:26 EST 2000


Hi Indira,
Sound like a problem in transfection efficiency. Several reasons are possible:
* your transfection reagent might have gone bad. Try new reagent or a
different method of transfection (superfect, electroporation, polyethyleneimine)
* your DNA to be transfected is damaged/degraded, or of poor purity.
Check an aliquot on an agarose gel. If the DNA looks ok, use a different
method for plasmid purification. Classical CsCl-banding is still the
best, but you can also use those commercial kits as long as you make
sure you don´t overload the columns (never use more than 60% of the
bacterial culture volume stated in the manuals!)
* your cells might suffer from either poor serum quality or mycoplasma
infection. Perform a sensitive mycoplasma assay and discard infected
cells. If this assay is negative, check a different batch of serum.

Hope this helps,
Frank


IndiraShukla wrote:
> 
> Hi, I will appreciate any input in the following problem.
> 
> I am trying to express a 10kD protein which is fused to GFP protein
> (27kD). I was able to have very good expresion only ONCE. I have not been
> able to repeat the expression again. Even GFP alone does not express very
> well. I am using AtT20 cells. We had a similar problem with GH3 cells. As
> far as I know, I am doing everything the same as I did that one time
> except the cell density, i did not count it that time, but I have tried
> varying degree of confluency with no success. Adding Sodium butyrate does
> not help. Can anyone with experience in expressing proteins in mammalian
> cells give me soem pointers.
> 
> Thanks.






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